Heartworm and Wolbachia: Therapeutic implications

Heartworm and Wolbachia: Therapeutic implications

J.W. McCall a,*, C. Genchi b, L. Kramer c, J. Guerrero d, M.T. Dzimianski a,P. Supakorndej a, A.M. Mansour e, S.D. McCall e, N. Supakorndej e,

G. Grandi e, B. Carson e

a Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA, United Statesb Dipartimento di Patologia Animale, Igiene e Sanita Pubblica Veterinaria, Sezione di Patologia Generale e Parasitologia,

Universita degli Studi di Milano, Milano, Italyc Dipartimento de Produzione Animali, Universita di Parma, 43100 Parma, Italy

d Department of Pathobiology, School of Veterinary Medicine, 3800 Spruce Street, University of Pennsylvania, Philadelphia, PA, United Statese TRS Labs, Inc., 295 Research Drive, Athens, GA, United States

Abstract

A safer, more effective adulticidal treatment and a safe method for reducing microfilaremia and breaking transmission of

heartworm disease early in the treatment are needed. The present study evaluated efficacy of ivermectin (IVM) and doxycycline

(DOXY) alone or together (with or without melarsomine [MEL]) in dogs with induced adult heartworm infection and assessed the

ability of microfilariae from DOXY-treated dogs to develop to L3 in Aedes aegypti mosquitoes and subsequently to become

reproductive adults in dogs. Thirty beagles were each infected with 16 adult heartworms by intravenous transplantation. Six weeks

later, dogs were ranked by microfilarial count and randomly allocated to 6 groups of 5 dogs each. Beginning on Day 0, Group 1

received IVM (6 mcg/kg) weekly for 36 weeks. Group 2 received DOXY (10 mcg/(kg day)) orally Weeks 1–6, 10–11, 16–17, 22–

25, and 28–33. Groups 3 and 5 received IVM and DOXY according to doses and schedules used for Groups 1 and 2. At Week 24,

Groups 3 and 4 received an intramuscular injection of MEL (2.5 mg/kg), followed 1 month later by two injections 24 h apart. Group

6 was not treated. Blood samples were collected for periodic microfilaria counts and antigen (Ag) testing (and later immunologic

evaluation and molecular biology procedures). Radiographic and physical examinations, hematology/clinical chemistry testing, and

urinalysis were done before infection, before Day 0, and periodically during the treatment period. At 36 weeks, the dogs were

euthanized and necropsied for worm recovery, collection of lung, liver, kidney, and spleen samples for examination by

immunohistochemistry and conventional histological methods.

All dogs treated with IVM + DOXY (with or without MEL) were amicrofilaremic after Week 9. Microfilarial counts gradually

decreased in dogs treated with IVM or DOXY, but most had a few microfilariae at necropsy. Microfilarial counts for dogs treated

only with MEL were similar to those for controls. Antigen test scores gradually decreased with IVM + DOXY (with or without

MEL) and after MEL. Antigen scores for IVM or DOXY alone were similar to controls throughout the study. Reduction of adult

worms was 20.3% for IVM, 8.7% for DOXY, 92.8% for IVM + DOXY + MEL, 100% for MEL, and 78.3% for IVM + DOXY.

Mosquitoes that fed on blood from DOXY-treated dogs had L3 normal in appearance but were not infective for dogs.

Preliminary observations suggest that administration of DOXY + IVM for several months prior to (or without) MEL will

eliminate adult HW with less potential for severe thromboembolism than MEL alone.

# 2008 Elsevier B.V. All rights reserved.

Keywords: Canine heartworm; Dirofilaria immitis; Ivermectin; Doxycycline; Melarsomine dihydrochloride; Wolbachia

www.elsevier.com/locate/vetpar

Available online at www.sciencedirect.com

Veterinary Parasitology 158 (2008) 204–214

* Corresponding author. Tel.: +1 706 542 5684/+1 706 542 8449; fax: +1 706 542 5771.

E-mail address: [email protected] (J.W. McCall).

0304-4017/$ – see front matter # 2008 Elsevier B.V. All rights reserved.

doi:10.1016/j.vetpar.2008.09.008

mailto:[email protected]

http://dx.doi.org/10.1016/j.vetpar.2008.09.008

J.W. McCall et al. / Veterinary Parasitology 158 (2008) 204–214 205

Table 1

Timing of treatments and evaluations in dogs with induced infections

with adult Dirofilaria immitis.

Treatment Timing of treatments

and evaluations

Ivermectin Weeks 1–36

Doxycycline Weeks 1–6, 10–11, 16–17,

22–25, 28–33

Ivermectin+ Weeks 1–36

Doxycycline+ Weeks 1–6, 10–11, 16–17,

22–25, 28–33

Melarsomine Week 24, 28a/ba

Melarsomine Weeks 24, 28a/ba

Ivermectin+ Weeks 1–36

Doxycycline Weeks 1–6, 10–11, 16–17,

22–25, 28–33

Control No treatment

Evaluations

Microfilarial counts/Ag Weeks –6, 0, 6, 10, 12, 16,

18, 24, 26, 28, 32, 36

Radiographs Weeks –6, 0, 12, 24, 28, 32, 36

Hematology/clinical pathology

/physical exam/urinalysis

Weeks 0, 12, 24, 28, 32, 36

a Two injections of melarsomine were administered 24 h apart at

Week 28.

1. Introduction

All current evidence suggests that Wolbachia (Sironi

et al., 1995), which are bacteria found in all individuals

of the filarial species that are known to harbor these

bacteria, are endosymbionts (i.e., the bacteria is

essential for the filarial worms’ survival) (Taylor

et al., 2005a). Tetracyclines inhibit the early develop-

ment of filarial infections in animal models and block

embryogenesis in human filariae and heartworms,

presumably by elimination of the Wolbachia from the

worm (Bosshardt et al., 1993; Bandi et al., 1999;

Hoerauf et al., 1999, 2001, 2000a,b, 2003a,b; McCall

et al., 1999; Langworthy et al., 2000; Townson et al.,

2000; Genchi et al., 2001; Casiraghi et al., 2002; Rao

and Weil, 2002; Rao et al., 2002; Chirgwin et al., 2003;

Volkmann et al., 2003; Taylor et al., 2005a,b; Smith and

Rajan, 2000). In contrast, filarial species that do not

harbor Wolbachia are not affected by tetracyclines

(Hoerauf et al., 1999; McCall et al., 1999). Wolbachia-

derived proteins have been implicated in the inflam-

matory response (Brattig et al., 2001, 2004; Cross et al.,

2001; Taylor et al., 2001; Bazzocchi et al., 2003; Grandi

et al., 2005; Hise et al., 2004; Kramer et al., 2005),

innate and adaptive immune responses (Bazzocchi

et al., 2000, 2007; Punkosdy et al., 2003; Simon et al.,

2003; Brattig et al., 2004; Kramer et al., 2003, 2005;

Morchon et al., 2004) and tolerized immunological

phenotype (O’Connor et al., 2003; Brattig et al., 2004;

Turner et al., 2006; Morchon et al., 2007a,b) associated

with human filariasis and heartworm disease in dogs.

Protracted monthly prophylactic doses of ivermectin

(IVM) provide ‘‘safety-net’’ activity against developing

heartworms and a ‘‘slow-kill’’ effect on adult heart-

worms, with earlier inhibition of embryogenesis

(McCall, 2005; McCall et al., 1995, 1996, 1998,

2001). The unique drug effects of IVM on 3–8-month-

old heartworms are related to the age of the worms at

initiation of treatment. The earlier treatment is started,

the shorter the survival time of the worms and the more

stunted are the worms. Worms that are fully grown

when treatment is started are not shorter after prolonged

monthly treatment, but worm mass is reduced by at least

20% due to the death of all uterine stages of

microfilariae and the ‘‘wasting away’’ condition of

the worms (McCall, 1993, unpublished data).

Melarsomine dihydrochloride (MEL) is the only

drug approved in the United States by the Food and

Drug Administration as an adulticide for heartworms.

Two intramuscular injections (2.5 mg/kg given 24 h

apart) or three injections (2.5 mg/kg followed 1-month

later by two injections 24 h apart) are the two treatment

protocols available for MEL. The American Heartworm

Society (AHS) recommends the three-injection protocol

because it is safer and more efficacious. The AHS

further indicates that it may be beneficial to administer a

macrocyclic lactone preventative for up to 6 months

prior to the adulticide, when the presentation does not

demand immediate attention (Nelson et al., 2005).

Death of adult filarial worms, whether due to treatment

or natural causes, results in an exacerbated inflammatory

response. We hypothesize that this heightened response is

due in part to the release of Wolbachia following death

and disintegration of the worms.

The study presented here describes an evaluation of

the efficacy of IVM and DOXY, alone or together (with

or without MEL) in dogs with induced, adult infection

with Dirofilaria immitis and assessment of the ability of

microfilariae from DOXY-treated dogs to develop to 3rd

stage in A. aegypti mosquitoes and then to become

reproductive adults in dogs. This article is part of a

complex study that is still ongoing and other data from

the study is being published.

2. Materials and methods

2.1. Study design

An overview of the study design for, 30-dog

experiment and is presented in Table 1.

J.W. McCall et al. / Veterinary Parasitology 158 (2008) 204–214206

2.2. Animals

A total of 30 purpose-bred beagles, including 23

males and 7 females, 12–16.5 months of age, weighing

15.1–34.2 kg, were used in the study. The dogs were

housed in mosquito-proof indoor pens in a purpose-built

building, with controlled temperature and ventilation

systems. The dogs were fed at least once daily an

appropriate quantity of commercially available main-

tenance diet and water was supplied ad libitum. Treated

dogs were housed individually in runs, and nontreated

dogs were housed 1–3 of the same sex to a run. The

animals were maintained with due regard for their

welfare and in accordance with applicable laws,

regulations and guidelines. The protocol was approved

by the Institutional Animal Care and Use Committee

prior to initiation of the study.

An additional 11 heartworm-naive beagles were

obtained from the commercial supplier for the mosquito

studies (M-1, M-2, and M-3). Two of these dogs were

experimentally infected with heartworms, became

microfilaremic, and were used as donors in study M-

3. The remaining 9 dogs were used as recipients in the

mosquito studies.

All personnel making observations, performing tests

and procedures, and collecting data were blinded in

regard to which were treated and control animals.

Groups were color-coded for identification by labora-

tory personnel throughout the study.

2.3. Parasite

Each of the 30 dogs was infected with nine adult

female and seven adult male heartworms by intravenous

transplantation via a jugular vein (Dzimianski et al.,

1989). The heartworms ranged in age from 10 to 21

months and represented three different laboratory

strains of the parasite (Butch, Lady, and Missouri

strains).

2.4. Dog treatment study

Approximately 6 weeks after infection, the 30 dogs

were ranked by microfilariae count within gender, and

then randomly allocated to a nontreated control group of

five dogs and five test groups of five dogs each. Each

group contained at least one dog of each sex.

2.5. Study drugs

Commercially available chewables (Heartgard1

Plus, Merial Limited, Duluth, GA) containing IVM

and pyrantel pamoate were administered orally accord-

ing to instructions on the label, with IVM given at a

minimum dosage of 6 mcg/kg of body weight and

pyrantel given at a minimum dosage of 5 mg/kg of body

weight.

Doxycycline hyclate (West-ward Pharmaceuticals,

Inc., Eatontown, NJ) was given orally for varying

periods throughout the study at a dosage equivalent to

10 mg doxycycline/(kg day). Melarsomine dihy-

drochloride (Immiticide1, Merial Limited, Duluth,

GA) was administered as intramuscular injections in the

epaxial muscles at 2.5 mg/kg of body weight per

injection. The three-injection protocol (alternate sche-

dule) was used: one injection followed 1 month later by

two injections 24 h apart.

All treatments with IVM and DOXY were started

on Day 0 (Table 1). Group 1 dogs were given IVM

weekly for 36 weeks. Group 2 dogs were given DOXY

daily during Weeks 1–6, 10–11, 16–17, 22–25, and

28–33. Group 3 dogs were given both IVM plus

DOXY as for Groups 1 and 2 and MEL (IVM + DOX-

Y + MEL) as for Group 4. Group 4 dogs were given

one injection of melarsomine (MEL) at 24 weeks,

followed 1 month later by 2 injections 24 h apart.

Group 5 dogs were given IVM plus DOXY as for

Groups 1 and 2. Group 6 served as the nontreated

control.

Whole blood and serum samples were collected for

microfilariae and D. immitis antigen (Ag) testing,

immunologic evaluation and molecular biology proce-

dures before IVM and DOXY dosing was initiated and

again at Weeks 6, 10, 12, 16 (microfilariae/Ag only), 18

(microfilaria/Ag only), 24, 26 (microfilariae/Ag only),

28, 32, and 36. Radiography, physical examinations,

hematology/clinical chemistry testing and urinalysis

were performed before infection (radiography only),

before Day 0, and again at Weeks 12, 24, 28 (Groups 3

and 4), 32 (Groups 3 and 4) and 36 (Table 2). All of the

dogs were humanely euthanized and necropsied at 36

weeks.

2.6. Microfilarial counting and antigen testing

At each blood collection, a 20-mL sample was

removed to make a Giemsa-stained preparation

(Schlotthauer et al., 1986), and 1 mL was retained

for later examination by the modified Knott method. For

the modified Knott method, the 1-mL samples were

mixed with 2% formalin and kept until after the 20-mL

slide preparations were examined. If microfilariae were

not seen on this preparation, the entire 1-mL sample was

examined.


Table 2

Efficacy of ivermectin and doxycycline, given alone or together with or without melarsomine, beginning 6 weeks after infection at necropsy 36

weeks after intravenous transplantation of live adult Dirofilaria immitis.

Treatment Mean no. live adult heartworms Percent efficacy

Male Female Total Range

Weekly ivermectin (6 mg/kg) 4.2a 6.8a 11.0a 5–14 20.3

Intermittent doxycycline (10 mg/(kg day)) 4.6a 8.0a 12.6a 9–15 8.7

Weekly ivermectin + intermittent

doxycycline + melarsomine (three injections)

0.8b 0.2c 1.0b,c 0–4 92.8

Melarsomine (three injections) 0b 0c 0c – 100

Weekly ivermectin + intermittent doxycycline 0.4b 2.6b 3.0b 0–6 78.3

Nontreated control 5.4a 8.4a 13.4a 12–16 NA

Different superscripts in a column indicate a significant difference between groups ( p � 0.01), NA = not applicable.

At each sampling, whole blood samples were

collected in EDTA for later use, and serum was

collected and stored at�70 8C until needed. Serum was

examined for adult D. immitis Ag using a commercially

available kit (DiroCHEK1, Symbiotics Corporation,

San Diego, CA). A subjective scoring system, based on

the intensity of the color reaction, was used to express

the results of each test as negative (�, 0); very weak

positive (vwk+, 1), weak positive (wk+, 2); positive (+,

3); strong positive (++, 4); or very strong positive (+++,

5) (McCall et al., 1996).

2.7. Necropsy

Just prior to necropsy, each dog was given

approximately 2 mL of heparin (1000 USP units/mL)

intravenously and the appropriate dose of a barbiturate

euthanasia solution. The heart and lungs were examined

grossly for pathological changes. The pleural and

peritoneal cavities were examined for D. immitis and the

anterior and posterior venae cavae were clamped before

removal of the heart and lungs. The right atrium, right

ventricle, and pulmonary arteries (including those

coursing through the lungs) were dissected and

examined for worms. The worms from each dog were

recorded as either dead or alive; live worms were further

classified by motility and appearance compared with

those from control dogs. Worms were then sexed and

counted. For later studies, some female worms from

each dog were dissected and the removed intestines and

reproductive tracts were placed in EM fixative, some

entire worms were placed in EM fixative, some worms

were frozen, some were placed in RNA later, and some

were placed in 10% formalin. Also for later studies,

lung, liver, kidney, and spleen samples were taken from

each dog for examination by immunohistochemistry,

conventional microscopy, transmission electron micro-

scopy and molecular biology techniques.

2.8. Statistical analysis

The numbers of live male worms, live female worms,

and total live worms were determined for each dog. The

numbers of worms recovered for each dog were

transformed to the natural logarithm (count + 1) for

calculation of geometric means and analysis, using one-

way analysis of variance for complete randomized

design. Each group was compared with the control

group, using a single degree of freedom contrast.

Groups were compared with each other using Duncan’s

multiple range test. Efficacy against male, female, and

total worms in each test group was calculated as the

percent reductions, using geometric means, compared

with the control group.

2.9. Mosquito studies

For M-1 and M-2 experiments, pooled blood samples

from several dogs in each of Groups 1 (IVM only), 2

(DOXY only), and 6 (nontreated control) were

membrane-fed to adult female A. aegypti (Liverpool

Black-eyed strain) mosquitoes (McCall, 1981). Sixteen

days later, L3 from each of the three sets of mosquitoes

were collected in approximately 1 mL of Hanks’

balanced salt solution (pH 7.0) containing penicillin

(0.4 units/mL) and streptomycin (0.4 mg/mL) and

injected subcutaneously into the inguinal area of each

of three dogs using a tuberculin syringe with a 20-gauge

needle.

For the M-3 experiment, blood from a donor dog

treated only with DOXY (Lady strain) as for M-1 and

blood from a donor control dog (Lady strain) were

membrane-fed to two groups of mosquitoes. Infective-

stage larvae from the DOXY mosquitoes were injected

subcutaneously into two recipient dogs, and one

recipient dog was given L3 from the mosquitoes fed

on the blood from the donor control dog.


Fig. 1. Mean microfilarial counts for dogs given weekly ivermectin

(IVM) or intermittent doxycycline (DOXY), alone or together (with or

without melarsomine [MEL] later), beginning 6 weeks after intrave-

nous transplantation of adult heartworms.

The mosquitoes for the M-1 experiment were

infected with microfilaremic blood on Day 66 (Week

9, 3 days after the last dose of IVM and 25 days after the

last dose of DOXY). Sixteen days later, the total of six

L3 collected from the IVM mosquitoes were injected

subcutaneously into Recipient dog 1, 40 of the L3

collected from the DOXY mosquitoes were injected

subcutaneously into Recipient dog 2, and 40 of the L3

collected from the control mosquitoes were injected

into Recipient dog 3. Microfilariae and Ag testing was

performed 5.8, 6.7, 7.1, 8.0, 8.8, 9.4, and 10.4 months

after infection and the three dogs were necropsied for

recovery of adult worms at 10.8 months.

The mosquitoes for the M-2 experiment were

infected on Day 129 (Week 18). At this point in the

study (i.e., 3 days after the last dose of IVM and 4 days

after the last dose of DOXY), microfilariae counts for

the IVM- and DOXY-treated groups had already

decreased substantially. The total (3) L3 collected from

the IVM mosquitoes were injected into Recipient dog 4;

the total (5) L3 collected from the DOXY mosquitoes

were injected into Recipient dog 5; and 40 of those from

the control mosquitoes were injected into Recipient dog

6. Microfilaria and Ag testing was done at 5.0, 5.9, 6.7,

7.3 and 8.3 months after infection and the dogs were

necropsied 8.7 months after infection.

For the M-3 experiment, the mosquitoes were

infected with microfilaremic blood on Day 54 (Week

8, 14 days after the last dose of DOXY). Sixteen days

later, Recipient dogs 7 and 8 were each injected with 40

L3 larvae from the DOXY mosquitoes and Recipient

dog 9 received 40 control larvae. Recipient dog 7 was

necropsied 4.2 months after infection, which was too

early for microfilariae and Ag testing. Microfilariae and

Ag tests were performed for the two remaining

Recipient dogs (8 and 9) just prior to necropsy at 6

months after infection.

All dogs infected by either intravenous transplanta-

tion or subcutaneous injection of L3 were negative for

heartworm microfilariae and Ag prior to infection.

3. Results

3.1. Dog treatment study

Weekly administration of IVM plus intermittent

DOXY (IVM + DOXY) for 36 weeks was 78.3%

effective in reducing the adult worm burden

(Table 2). One of the five dogs in this group was

completely cleared of worms, and most of the

remaining worms in the other four dogs were abnormal

in appearance, with intermittent translucent areas in the

body and dark anterior ends. In comparison, treatment

with only IVM was only 20.3% effective. All dogs in

this group had adult worms, and most of the worms were

abnormal in appearance, with intermittent translucent

areas in the body and dark anterior ends. Treatment with

only DOXY was only 8.7% effective. Treatment with

IVM + DOXY + MEL was 92.8% effective against

adult worms. Three of the five dogs in this group were

cleared of worms, and the two remaining dogs had 1

male and 4 (3 males, 1 female) live worms, respectively.

All of these worms were abnormal in appearance. MEL

alone was 100% effective in reducing the worm burden;

one dead female worm was recovered from one dog and

four of the five dogs had fragments of worms. In

comparison, all nontreated control dogs had live worms,

with an average of 13.4 per dog (range = 12–16). All

except two of the worms from these nontreated control

dogs were normal in appearance.

3.1.1. Microfilarial counts

Group mean microfilarial counts are presented in

Fig. 1. All dogs were negative for circulating

microfilariae prior to infection. All dogs also were

positive for microfilariae just prior to Day 0, except

one dog, which became microfilaremic by Week 6. For

both groups that were given IVM plus DOXY (with or

without MEL), mean microfilariae counts dropped

relatively soon after treatment started, and no

microfilariae were seen in any of these 10 dogs after

Week 9. Beginning approximately Week 10, mean

microfilariae counts for dogs that received IVM + -

DOXY dropped gradually to negative or near-negative

values (generally � 10 microfilariae/mL) just prior to

necropsy. At necropsy, all five DOXY dogs had

microfilariae. Two of the five dogs treated with IVM

were amicrofilaremic, and three of them had 2, 4, and

100 microfilariae/mL, respectively, just prior to


necropsy at 36 weeks. Mean microfilariae counts in

the MEL and nontreated control groups remained

relatively high throughout the study.

3.1.2. Antigen scores

Group mean Ag scores for the nontreated control

and treated groups are presented in Fig. 2. Although the

score for the IVM + DOXY + MEL group spiked at

Week 32, the scores for both groups that received IVM

plus DOXY (IVM + DOXY and IVM + DOXY + -

MEL) gradually dropped after Week 26, and the score

for both groups was 2.2 just prior to necropsy. The Ag

score for the MEL-treated group did not drop until just

prior to necropsy (score = 1.8). The mean scores for the

groups that received only IVM or DOXY were similar

to those for nontreated controls throughout the study

(range 3.8–4.2).

3.2. Mosquito studies

3.2.1. M-1 study

Recipient dog 3 that was given 40 infective larvae

from mosquitoes that had fed on blood from nontreated

dogs was positive for microfilariae and Ag 6.7 months

post-infection and remained positive on both tests

through to the time of necropsy at 10.8 months PI. At

necropsy, this dog had a total of 27 adult worms.

Recipient dog 1 that was given 6 infective larvae from

the mosquitoes that had fed on blood from the dogs

treated with IVM was negative for microfilariae and Ag

throughout the study and had two live adult male worms

at necropsy. Recipient dog 2 that received 40 infective

larvae from mosquitoes that had fed on blood from dogs

treated with DOXY was negative for microfilariae and

Ag throughout the study and had no live worms at

necropsy.

Fig. 2. Mean heartworm antigen test (DiroCHEK1) scores for dogs

administered weekly ivermectin (IVM) or intermittent doxycycline

(DOXY), alone or together (with or without melarsomine [MEL]

later), beginning 6 weeks after intravenous transplantation of adult

heartworms.

3.2.2. M-2 study

Recipient dog 6 that was given 40 larvae from

mosquitoes that had fed on blood from the nontreated

dogs was positive for microfilariae and Ag at 5.9 months

after infection and remained positive on both tests

through to the time of necropsy at 8.7 months after

infection. At necropsy, this dog had a total of 26 adult

worms. Recipient dog 5 that received five infective


treated with DOXY was negative for microfilariae and

Ag throughout the study and had no live worms at

necropsy. Recipient dog 4 that was given six infective


treated with IVM was negative for both microfilariae

and Ag throughout the study but had one live adult

female worm at necropsy.

3.2.3. M-3 study

No live worms were recovered from Recipient dog 7

necropsied 4.2 months after infection with 40 larvae

from mosquitoes that had fed on a dog treated with

DOXY. Just prior to necropsy at 6 months after

infection, Recipient dog 8, which was also infected with

larvae from mosquitoes that fed on a dog treated with

DOXY, was negative for microfilariae and Ag and had

no worms at necropsy. Control dog 9 was positive for

both microfilariae and Ag and had a total of 21 live adult

worms at necropsy.

4. Discussion

Weekly administration of prophylactic doses (6 mcg/

kg) of IVM resulted in a gradual reduction in

microfilarial count to negative (for two dogs) or near-

negative (2–100 microfilariae/mL) values by the end of

the study. These results are similar to those obtained in

other studies where IVM was given monthly at the same

dosage (McCall et al., 1998, 2001; Bowman et al.,

2001). Intermittent administration of daily doses of

DOXY caused a gradual reduction in microfilariae

count, with a pattern similar to that of the IVM-only

group, but all of the five dogs had a few microfilariae

(�10 microfilariae/mL) just prior to necropsy at 36

weeks. Starting treatment with DOXY and IVM

treatments approximately 6 months before the first

treatment with MEL was to clear the Wolbachia from

the worms as much as possible before initiating

treatment with MEL. In our previous laboratory studies,

we have seen a rebound in Wolbachia at varying times

after treatment. Giving intermittent treatments over the

course of the study was expected to preclude that

rebound.


These gradual reductions in microfilariae counts

during administration of IVM or DOXY only strongly

suggest that inhibition of embryogenesis (DOXY) and

death of developing microfilariae stages in utero (IVM)

led to microfilariae reduction due to attrition rather than

to a direct microfilaricidal effect of the drugs (Lok et al.,

1989; McCall et al., 2001; Taylor et al., 2005a). The

complete disappearance of circulating microfilariae

after Week 10 in all 10 dogs treated with DOXY + IVM

suggests that this combination of the drugs had a direct

microfilaricidal effect as well.

The lower mean Ag scores in the dogs that were

given MEL only, IVM + DOXY or IVM + DOXY + -

MEL generally reflected their lower worm burdens

(along with the presence or absence of worm fragments

as a source of Ag), compared with the other groups. The

gradual reduction in average Ag scores for the groups of

dogs that received IVM + DOXY (with or without

MEL) indicates that some of the worms died relatively

early during the treatment period and only a few worms

were alive at necropsy, as evidenced by the 78.3%

efficacy in the IVM + DOXY group and the 92.8%

efficacy for IVM + DOXY + MEL. The spike in Ag

score for the IVM + DOXY + MEL group at 32 weeks

was probably due to the release of Ag from worms

killed by MEL, and the dramatic reduction in Ag score

in the MEL group between Weeks 32 and 36 was

probably due to the death and clearance of Ag, since

efficacy was 100% for this group.

The 20.3% reduction in worm burden following

weekly administration of prophylactic doses of IVM for

about 8.5–9 months was not surprising. In an earlier

study, 16 monthly doses of the drug were only 56%

effective against adult heartworms (McCall et al.,

1998), which suggests that weekly dosing only with

IVM at the prophylactic dosage is no more effective in

killing adult heartworms than monthly dosing. The lack

of early adulticidal efficacy of DOXY alone against

heartworms is consistent with findings for other filariae,

which usually require at least 1.5–2 years for adulticidal

effects to be evident (Langworthy et al., 2000; Gilbert

et al., 2005; Taylor et al., 2005a, b). IVM + DOXY had

substantial (78.3%) adulticidal efficacy, and it seems

reasonable to assume that the remaining worms would

have died if the dogs had been held a few more months

before necropsy. This high level of adulticidal activity

of IVM + DOXY strongly suggests a synergistic effect

of these two drugs and warrants further attention. In the

current study, treatment with IVM and DOXY was

purposefully excessive in an attempt to obtain near

maximal effect. Follow-up studies will focus on

increasing the daily dosage of DOXY and shortening

the initial treatment period to 4 weeks, followed by

treatment 4–6 months later and administering IVM no

more frequently than every 2 weeks. Limiting DOXY

treatment to one period of 4–6 weeks has been effective

in treatment patients with human filariasis (Taylor et al.,

2005b).

The three-injection protocol for MEL was 100%

effective against adult heartworms, whereas IVM + -

DOXY + MEL was 92.8% effective. This might suggest

drug interference, however, MEL is not always 100%

effective (Dzimianski et al., 1989; Di Sacco and

Vezzoni, 1992; Keister et al., 1992; Vezzoni et al.,

1992). Confirmation of drug interference would require

a study with a larger dog population.

Population dynamics in relation to the role of

Wolbachia in various life-cycle stages has been studied

in filariae, particularly Brugia malayi (McGarry et al.,

2004; Fenn and Blaxter, 2004; Kozek, 2005; Taylor

et al., 2005a). Individual worms appear to vary widely

in their bacterial load, which may reflect a dynamic

change of population size over time or if constant, the

potential for a selective advantage in terms of longevity

or fecundity in worms carrying more bacteria. However,

at the population level, the numbers of bacteria remain

static in microfilariae and the mosquito-borne stages (L2

and L3), with the lowest ratios of Wolbachia/nematode

DNA. Within the first week of infection of the

mammalian host, bacteria numbers increase dramati-

cally and the bacteria/worm ratio is the highest of all

life-cycle stages. The rapid multiplication continues

throughout L4 development, so that the major period of

bacterial growth occurs within the first month of

infection. There are few bacteria in mosquito-derived L3

but many, in large groups, in L4 collected 1–3 weeks

after infection. It appears that the large clusters of

bacteria observed throughout the hypodermal cord of

adult worms originate from this rapid period of division

and thereafter are maintained at that level, as seen in

adult male worms. In females, bacterial numbers

increase further as the worms mature and as the ovary

and embryonic larval stages become infected. Thus,

population dynamics is consistent with (1) the two

processes compromised most by ‘‘tetracycline’’ treat-

ment (i.e., molts from L3–L4 and L4–juvenile; micro-

filariae production) and (2) evasion of mammalian

immunity (i.e., L3–L4 and L4–juvenile molts and adult

worm survival) allowing for long-term survival of adult

filariae. In the present study, the survival of micro-

filariae after DOXY treatment of HW-microfilaremic

dogs, followed by ‘‘normal’’ development of these

microfilariae to L3 and the inability of these L3 to

develop to adults in recipient dogs strongly suggest that


relatively few (if any) Wolbachia are required for

microfilariae to survive and develop into L3, but

substantial numbers are needed for development of

L3 into adults in the mammalian host.

Arsenical drugs have been the mainstay for heart-

worm adulticidal therapy for the past four to five

decades. However, treatment strategies have changed,

and indeed, continue to evolve since MEL replaced

thiacetsamide sodium in the early 1990s. The manu-

facturer provides two treatment protocols for MEL: the

‘‘standard’’ two-injection protocol for Class I (mild

clinical signs) and Class II (moderate clinical signs)

dogs and the ‘‘alternative’’ three-injection protocol for

Class III (severe clinical signs) disease. Generally, this

was followed 3–4 weeks later by administration of a

microfilaricide, which often had to be repeated once or

twice to clear the dog of circulating microfilariae.

During the past several years, this microfilaricidal

treatment generally has been reduced to a by-product of

macrocyclic lactone chemoprophylaxis (Nelson et al.,

2005).

Starting in 1995, several studies have demonstrated

that prolonged administration of monthly prophylactic

doses of a macrocyclic lactone, particularly IVM, kills

older larvae, immatures (juveniles), young adults and

mature adults (see McCall, 2005). Moreover, a high

percentage of the dogs become amicrofilaremic within

6–9 months after dosing is started. The rate of kill with

this slow-kill treatment is dependent on the age of the

heartworms when treatment is started, with 3-month-

old larvae requiring up to 1 year and mature adults

needing 2.5 years to provide efficacy of at least 95%.

Although monthly IVM is not an approved alternative to

MEL therapy and it is particularly risky in very active

and symptomatic dogs (Venco et al., 2004), it clearly

provides potent ‘‘safety-net’’ activity against older

larvae, immatures (juveniles), and young adults in cases

of owner compliance failure, even when the owner and

veterinarian are not aware that the animal is infected.

The American Heartworm Society (Nelson et al.,

2005) recognizes the safety-net and adulticidal proper-

ties of some of the macrocyclic lactones, particularly

IVM. Their 2005 canine guidelines state that admin-

istration of a chemoprophylactic dose of a macrocyclic

lactone should begin as soon as the dog is diagnosed

with a heartworm infection. While controversial due to

the theoretical risk of inducing resistance to macro-

cyclic lactones, it may be beneficial to administer a

macrocyclic lactone for up to 6 months prior to

administration of MEL when the presentation does not

demand immediate intervention. The reasoning for this

approach is to greatly reduce, if not completely

eliminate, circulating microfilariae and kill migrating

D. immitis larvae and (in the case of IVM) to stunt

immature D. immitis (�20%) (McCall, 1993, unpub-

lished data) and reduce female worm mass (�20%)

(McCall, 1993, unpublished data) by inhibiting the

reproductive system. Moreover, administration for 3

months also will allow immature worms to reach an age

at which they are known to be susceptible to killing by

MEL (Keister et al., 1992; Atkins and Miller, 2003) and

administration for longer than 3 months should result in

reduced antigenic mass, which in turn may reduce the

risk of thromboembolism.

By eliminating the Wolbachia endosymbionts,

tetracyclines have been shown in several species of

filariae to prevent development of the larval stages,

inhibit embryogenesis in adult worms, and eventually

kill adult filariae, usually 1.5–2 years after treatment is

started in laboratory animals, large animals, and

humans (Taylor et al., 2005a; Kramer et al., 2007).

In the present study, IVM + DOXY had the

synergistic effects of completely clearing dogs of

circulating microfilariae by Week 10 and killing 78.3%

of the adult worms by Week 36. Interestingly,

Wolbachia were still detected at necropsy in worms

from dogs treated only with DOXY but were virtually

eliminated in those receiving both DOXY and IVM

(Bazzocchi et al., 2008). Gross pathology and

histopathologic observations of the lungs of the 30

dogs revealed the most severe pathology in the dogs

receiving only MEL and the least evidence of pathology

in those receiving IVM + DOXY + MEL (no grossly

visible lesions in 3 of 5 dogs). These latter dogs also

showed significantly fewer severe arterial lesions and a

virtual absence of thrombi (Kramer et al., 2008).

Clinical findings of significance included eosinophilia

in most dogs treated with IVM or DOXY alone at 12

weeks following a precipitous decline in microfilariae;

only dogs that received IVM did not have elevations in

serum alanine transaminase; and MEL had no apparent

effect on serum chemistries (Dzimianski et al., 2006).

In the present study, L3 collected from mosquitoes

fed on microfilaremic blood from dogs treated with

DOXY were normal in appearance and motility but

were not able to develop in dogs. Thus, treatment with

DOXY prevented further transmission of the disease

even when microfilariae were present.

These and other observations strongly suggest that

administration of both IVM and DOXY for several

months prior to MEL or possibly without MEL, will

eliminate adult heartworms with less potential for

severe thromboembolism than MEL alone and will

block transmission. Therefore, it is likely that DOXY


eventually will be included in heartworm adulticide

therapy for dogs. Furthermore, the potentially life-

threatening infections and high risk associated with

MEL treatment strongly encourage the testing of

DOXY plus IVM as an alternative adulticidal therapy

for heartworm infected cats and ferrets (McCall et al.,

2008).

Conflict of interest

J.W. McCall, Genchi, L. Kramer, Guerrero, Dzi-

mianski, P. Supakorndej, Mansour, S.D. McCall, N.

Supakorndej, Grandi, and Carson have no personal or

financial relationship with other persons or organizations

that could inappropriately influence or bias this study.

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Heartworm and Wolbachia: Therapeutic implications